Post‐translationally modified neoantigens: Promising targets for diagnostic strategy of autoimmune diseases

Autoimmune diseases can be caused by emerging neoantigens that break immune tolerance in humans. With increasing prevalence of autoimmune diseases, the key understanding of emerging neoantigens and most importantly the related-diagnosis are urgently needed.1 The development of etiological diagnosis of autoimmune diseases is of great significance for disease targeted therapy. Asmajor histocompatibility complex (MHC) subtypes of patients are highly related to autoimmune disease, identifying newly appearing self-antigens that interact with MHC and induce adaptive immune responses is vital. Post-translational modifications (PTMs) such as phosphorylation, methylation and acetylation are known for their biological functions important for signal transduction and transcription regulation. PTMs have also been shown to


GENERATION OF POST-TRANSLATIONALLY MODIFIED NEOANTIGEN IN AUTOIMMUNE DISEASES
Autoimmune diseases can be caused by emerging neoantigens that break immune tolerance in humans. With increasing prevalence of autoimmune diseases, the key understanding of emerging neoantigens and most importantly the related-diagnosis are urgently needed. 1 The development of etiological diagnosis of autoimmune diseases is of great significance for disease targeted therapy.
As major histocompatibility complex (MHC) subtypes of patients are highly related to autoimmune disease, identifying newly appearing self-antigens that interact with MHC and induce adaptive immune responses is vital. Post-translational modifications (PTMs) such as phosphorylation, methylation and acetylation are known for their biological functions important for signal transduction and transcription regulation. PTMs have also been shown to This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. produce self-generated neoantigens from existing proteins and peptides by altering the primary structure of proteins, that is, by altering the protein 'self' sequence, without the need to introduce antigens from microbial and somatic mutations. One of possible source of PTM neoantigen is the changes of metabolites. The generation of metabolic PTM-related neoantigens in autoimmune diseases may be one of the main causes of disease and provide strategies for the systematic diagnosis and evaluation.
The PTM neoantigen-based autoimmune diseases diagnostic strategy mainly contains two parts: neoantigenbased disease risk assessment and disease activity assessment ( Figure 1).

F I G U R E 1
Post-translational modifications (PTMs) neoantigen-based clinical diagnostic strategy. Post-translationally modified neoantigens are generated from the chemical reaction on proteins, which changes the physicochemical properties of proteins, and then the PTM-related peptides are presented by human leukocyte antigen (HLA) molecules. The PTM neoantigens further active self-reactive T cell and promote autoantibody production. For the PTM neoantigen-based clinical diagnostic strategy, HLA haplotype, metabolite and PTM neoantigen tests are aimed to assess risk of autoimmune diseases; self-reactive T cell and autoantibody tests are to evaluate disease activity.

HLA haplotype assessment
Specific HLA haplotypes are highly related to autoimmune diseases. AS is one of the autoimmune diseases associated with some of MHC subtypes, such as HLA-B*27, HLA-DRB1*01, HLA-DRB1*04, HLA-DQB1*03 and HLA-DQB1*05. 3 Further studies showed the association of HLA-B*27 bound peptidomes 4 and TCR repertoire. 5 HLA haplotype assessments through gene sequencing or flow cytometry are used clinically, which has diagnostic value in predicting the autoim-munity of neoantigen and the severity of the related diseases.

Metabolite assessment
Metabolites are important in immune imbalance and crosstalk between host and pathogen. The changing metabolites could also be the metabolic substrates in chemical reactions for PTMs formation. 6 In AS, the higher level of 3-HPA can directly increase the level of post-translational modifications, which will generate more cysteine carboxyethylated neoantigens. Assessment of changed metabolites can reflect disease risk, since some metabolic substrate changes can directly affect posttranslationally modified peptide levels.

Post-translational modified neoantigen assessment
Post-translational modified neoantigen assessment is based on the detection of PTM proteins. Systematic screening of the PTM profile as well as metabolic profile of patients with autoimmune diseases is a way to assessment the levels of neoantigen generated by metabolite-induced modifications. However, clinically disease-related PTMspecific antibodies are potential reagents to test PTM proteins in blood and tissue samples from patients through immunoblotting, enzyme-linked immunosorbent assay and immunohistochemistry.

Neoantigen-specific self-reactive T cell assessment
The production and activation of self-reactive T cells is an important part in antigen-specific autoimmune damage. The T cell repertoire of patients with autoimmune diseases harbors self-reactive CD4+ T cells capable of inducing autoimmunity. 7 Different from HLA haplotype, metabolites and PTM-related antigen tests, neoantigenspecific self-reactive T cell assessment show more about the disease activities. Experimentally, it is hard to obtain the anti-TCR antibodies, which recognize specific MHCneoantigen complex. However, the mature MHC polymer platform enables the detection of antigen-specific selfreactive T cells in the researches of autoimmune disease and cancer. 8

Neoantigen-specific autoantibody assessment
The production of autoantibodies may be one of the hallmarks of disease activity. One of the clinically used autoantibody test is anti-citrullinated protein/peptide antibodies assay, which was reported in relation with rheumatoid factor in rheumatoid arthritis. 9 Another neoantigen-specific autoantibody assessment research have indicated that anti-cysteine carboxyethylated autoantibody is associated with ankylosing spondylitis. 2 Because autoantibodies are relatively easy to detect, they may be important candidates for disease activity testing. However, the selection of neoantigen-specific autoantibody standards should be cautious in order to pursue consistency in different diagnostic occasions.

A C K N O W L E D G E M E N T S
This work was supported by National Natural Science Foundation of China (grant number: 92169211) to Ping Zhu, National Natural Science Foundation of China (grant numbers: 82101885 and 82322029) to Yue Zhai.

C O N F L I F T O F I N T E R E S T S TAT M E N T
The authors declare no financial or commercial conflicts of interest.